Our research group proposes to investigate the mechanisms of ethanol pretreatment for preventing bioprosthetic heart valve calcification as a general model of the inhibition of pathologic calcification. The calcification of glutaraldehyde pretreated porcine aortic valve leaflets will be considered as a general model system for pathologic calcification. The discoveries during the previous program period demonstrating ethanol prevention of bioprosthetic leaflet calcification represent the most successful anticalcification strategy thus far brought forward by any research group. The working hypothesis of our proposal is that ethanol inhibits bioprosthetic leaflet calcification due to both lipid extraction, thus inhibiting the cell-membrane component of leaflet calcification, and alterations in collagen structure, which inhibit the characteristic collagen calcification noted in calcified leaflets. We will investigate this hypothesis through the following studies or experiments: l)It is hypothesized that ethanol alters collagen structure and this results in a conformational change unfavorable for mineralization. We will investigate the effects of ethanol pretreatment on collagen structure with model system studies of Type 1 collagen (rat tail tendon) and bioprosthetic heart valve leaflets. These studies will rely on comparisons between ethanol pretreated materials and appropriate controls utilizing biophysical techniques including FTIR, Raman spectroscopy, and circular dichroism. 2) It is also hypothesized that ethanol interacts with the conformational changes in collagen induced by glutaraldehyde, and that this may contribute to inhibition of bioprosthetic leaflet calcification. These Investigations will compare ethanol and control specimens of Type I collagen and bioprosthetic leaflets examining overall cross link content (native and glutaraldehyde), glutaraldehyde uptake and stability, and the overall assessment of conformational change associated with crosslinking. 3) We further hypothesize that ethanol exposure extracts lipids and proteins, and that this extraction process contributes to inhibition of the cell-oriented cuspal calcific deposits. Lipid and protein extraction may also affect the resulting collagen conformational change and other matrix alterations favorable to the inhibition of calcification. 4) We further hypothesize that noncollagenous protein binding contributes to the pathophysiology of bioprosthetic calcification, and that the matrix alterations and structural protein changes induced by ethanol exposure alter the endogenous activity, exogenous binding, and mechanistic effects of noncollagenous proteins, such as alkaline phosphatase and osteopontin, to the pathogenesis of bioprosthetic heart valve calcification.